The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art in the present invention.
Messenger RNA (mRNA), encoding physiologically important proteins for therapeutic applications, has shown significant advantages over DNA-based plasmid and viral vectors for delivering genetic material. The most important of these advantages are:                (i) high level of safety (reduces potential genome damage from viral or plasmid integration),        (ii) mRNA delivery results in immediate protein expression (unlike delayed responses that generally occur with plasmids),        (iii) mRNA allows for robust dose-dependent control over expression of proteins and        (iv) the simplicity of large scale synthesis of mRNAs compared to manufacturing of plasmid and viral vectors.        
Messenger RNAs can be encoded for virtually any known protein and can be delivered to specific tissues and organs by a variety of methods known to those skilled in the art. Once delivered, these mRNAs direct ribosomal protein expression within targeted tissues resulting in the production of many hundreds of proteins per mRNA molecule.
Several structural elements, present in each active mRNA molecule, are utilized to translate the encoded proteins efficiently. One of these elements is a Cap structure on the 5′-end of mRNAs, which is present in all eukaryotic organisms (and some viruses). Naturally occurring Cap structures comprise a ribo-guanosine residue that is methylated at position N7 of the guanine base. This 7-methylguanosine (7mG) is linked via a 5′- to 5′-triphosphate chain at the 5′-end of the mRNA molecule. Throughout this application, 7m and m7 are used interchangeably with equivalent meaning. The presence of the 7mGppp fragment on the 5′-end is essential for mRNA maturation, it:                protects the mRNAs from degradation by exonucleases,        facilitates transport of mRNAs from the nucleus to the cytoplasm and        plays a key role in assembly of the translation initiation complex (Cell 9:645-653, (1976); Nature 266:235, (1977); Federation of Experimental Biologists Society Letter 96:1-11, (1978); Cell 40:223-24, (1985); Prog. Nuc. Acid Res. 35:173-207, (1988); Ann. Rev. Biochem. 68:913-963, (1999); J. Biol. Chem. 274:30337-3040, (1999)).        
Only those mRNAs that carry the Cap structure are active in Cap dependent translation; “decapitation” of mRNA results in an almost complete loss of their template activity for protein synthesis (Nature, 255:33-37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); and Proc. Natl. Acad. Sci. USA, 72:1189-1193, (1975)).
Another element of eukaryotic mRNA is the presence of 2′-O-methyl nucleoside residues at transcript position 1 (Cap 1), and in some cases, at transcript positions 1 and 2 (Cap 2). The 2′-O-methylation of mRNA is required for higher efficacy of mRNA translation in vivo (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves nuclease stability of the 5′-capped mRNA. The mRNA with Cap 1 (and Cap 2) is a distinctive mark that allows cells to recognize the bona fide mRNA 5′ end, and in some instances, to discriminate against transcripts emanating from infectious genetic elements (Nucleic Acid Research 43: 482-492 (2015)).
A primary mRNA transcript carries a 5′-triphosphate group (5′-pppmRNA) resulting from initiation of RNA synthesis starting with NTP (typically GTP) in vivo. Conversion of 5′-triphosphorylated end of mRNA transcript into a Cap structure (Cap 0) occurs via several enzymatic steps (J. Biol. Chem. 250:9322, (1975); J. Biol. Chem. 271:11936, (1996); J. Biol. Chem. 267:16430, (1992)). These enzymatic reactions steps include:                Step 1: RNA triphosphatase converts 5′-triphosphate of mRNA to a 5′-diphosphate, pppN1(pN)x→ppN1(pN)x+ inorganic phosphate;        Step 2: RNA guanyltransferase uses GTP to transfer a GMP residue to the 5′-diphosphate of the mRNA, ppN1(pN)x+GTP→G(5′)ppp(5′)N1(pN)x+ inorganic pyrophosphate; and        Step 3: guanine-7-methyltransferase, uses S-adenosyl-methionine (AdoMet) as a cofactor and transfers the methyl group from AdoMet to the 7-nitrogen of the guanine base, G(5′)ppp(5′)N1(pN)x+ AdoMet→7mG(5′)ppp(5′)N1(pN)x+ AdoHyc).The RNA that results from these enzymatic activities is referred to as “5′ capped RNA” or “capped RNA”, and the combination of enzymes involved in this process that results in formation of “capped RNA” are referred to as “capping enzymes”. Capping enzymes, including cloned forms of such enzymes, have been identified and purified from many sources and are well known in the art (Prog. Nucleic Acid Res. Mol. Biol. 66:1-40, (2001); Prog. Nucleic Acid Res. Mol. Biol. 50:101-129, (1995); and Microbiol. Rev. 44:175, (1980)). The capped RNA that results from the addition of the cap nucleotide to the 5′-end of primary RNA by capping enzymes has been referred to as capped RNA having a “Cap 0 structure” (J. Biol. Chem. 269:14974-14981, (1994); J. Biol. Chem. 271:11936-11944, (1996)). Capping enzymes have been used to synthesize capped RNA having a Cap 0 structure in vitro (J. Biol. Chem. 255:11588, (1980); Proc. Natl. Acad. Sci. USA 94:9573, (1997); J. Biol. Chem. 267:16430, (1992); J. Biol. Chem. 269:14974, (1994); and J. Biol. Chem. 271:11936, (1996)).        
Capped RNA having a 5′-Cap 0 structure can be further transformed in vivo to a “Cap 1” structure by the action of (nucleoside-2′-O—) methyltransferase (J. Biol. Chem. 269:14974-14981, (1994); J. Biol. Chem. 271:11936-11944, (1996); and EMBO 21:2757-2768, (2002)). For example, vaccinia mRNA (nucleoside-2′-O) methyltransferase can catalyze methylation of the 2′-hydroxyl group of the 5′-penultimate nucleotide of 5′-capped RNA having a Cap 0 structure by the following reaction:7mG(5′)ppp(5′)N1pN2(pN)x+AdoMet→7mG(5′)ppp(5′)N12′-OMepN2(pN)x+AdoHyc.
Dimethylated capped RNAs having a Cap 1 structure have been reported to be translated more efficiently than capped RNAs having a Cap 0 structure (Nucleic Acids Res. 26:3208, (1998)). Eukaryotic cells utilize another (nucleoside-2′-O) methyltransferase (for example hMTR2 in human cells (Nucleic Acids Res. 39:4756 (2011)) to catalyze methylation of the 2′-hydroxyl group of the second transcribed nucleotide of 5′-capped RNA to convert the Cap 1 structure to a Cap 2 structure by the following reaction:7mG(5′)ppp(5′)N12′-OMepN2(pN)x+AdoMet→7mG(5′)ppp(5′)N12′-OMepN22′-OMe(pN)x+AdoHyc.Approximately 50% of eukaryotic mRNAs have a Cap 2 structure.
In order to produce long functional RNAs for various biological studies, a method of in vitro enzymatic synthesis of primary RNA was developed in the mid-1980s (Methods Enzymol. 180:51-62, (1989); Nucl. Acids Res., 10:6353-6362, (1982); Meth. Enzymol., 180:42-50 (1989); Nucl. Acids Res., 12:7035-7056, (1984) and Nucleic Acid Research 15: 8783-8798, (1987)).
After in vitro transcription, the primary mRNA transcript carrying 5′-triphosphate group can be further capped with capping enzymes. However, in vitro enzymatic 5′-capping is expensive, laborious, inefficient and difficult to control.
In view of these disadvantages, another method was developed for the in vitro synthesis of capped mRNA where a chemically synthesized dinucleotide 7mG(5′)ppp(5′)G (also referred to as mCAP) is used to initiate transcription (RNA 1: 957-967, (1995)). The mCAP dinucleotide contains the 5′-Cap 0 structure of mature mRNA but does not have 2′-O-methyl nucleosides characteristic for Cap 1 and Cap 2 structures.
However, there are two main disadvantages attributed to initiation of in vitro transcription using synthetic mCAP dinucleotide. The first is a strong competition of mCAP and pppG for initiation of mRNA synthesis. When mRNA is initiated with pppG the resultant ppp-mRNA is inactive in translation and immunogenic due to the presence of the 5′-triphosphate. Correspondingly, when mRNA is initiated with mCAP the resultant 5′-capped-mRNA is active in translation and is not as immunogenic.
In order to improve the ratio of 5′-capped to 5′-uncapped (or 5′-triphosphorylated; pppmRNA) mRNAs, an excess of 7mGpppG over pppG (from 4:1 to 10:1) must be used to favor the production of the 5′-Cap structure mRNA transcripts (up to 80-90%). The negative side of this approach is a significant reduction of overall yield of mRNA due to a fast depletion of GTP supply during transcription and a requirement for large quantities of a synthetic mCAP dimer which can be expensive. After transcription an additional treatment of crude mixture, containing both 5′-capped mRNA and 5′-pppmRNA, with alkaline phosphatase is necessary to remove uncapped 5′-triphosphate groups from pppmRNA in order to reduce immunogenicity of synthesized mRNA. The uncapped 5′-OH form of mRNA obtained after phosphatase treatment is inactive and does not participate in translation process.
Another disadvantage, a bi-directional initiation, can arise when using a non-symmetrical mCAP dinucleotide. There is a tendency of the 3′-hydroxyl group of either the G or 7mG moiety of 7mGpppG to serve as initiation point for transcriptional elongation with a nearly equal probability. It typically leads to a synthesis of two isomeric RNAs of the form 7mG(5′)pppG(pN)n and G(5′)ppp7mG(pN)n, in approximately equal proportions, depending on conditions of the transcription reaction (RNA 1: 957-967, (1995)).
To eliminate bi-directional initiation of mRNA synthesis with mCAP dinucleotide a novel modified mCAP analog in which the 3′-OH group of 7mG residue is replaced with OCH3 (“OMe”):7mG(3′-O-Me)pppG (also known as Anti-Reverse Cap Analog (ARCA)) was developed. ARCA initiates mRNA synthesis only in the correct forward orientation (RNA 7:1486-1495 (2001)). Several types of ARCA analogs are known in the art (see, for example, U.S. Pat. No. 7,074,596). However, a large molar excess of ARCA over pppG is still required to ensure that most mRNA transcript molecules have the 5′-Cap structure. A further disadvantage is that an mRNA with a Cap 1 structure cannot be synthesized using a 7mGpppG2′-OMe Cap dimer (RNA 1: 957, (1995)) or its ARCA analog.
Presently, the known routes to production of active long mRNAs containing a Cap 1 structure consist of enzymatic capping and enzymatic 2′ O-methylation of the 5′-triphosphorylated mRNA transcript or enzymatic 2′-O-methylation of mCAP-capped or ARCA-capped mRNA precursor (Nucleosides, Nucleotides, and Nucleic Acids, 25:337-340, (2006) and Nucleosides, Nucleotides, and Nucleic Acids 25(3):307-14, (2006)). Both approaches are quite laborious, difficult to control and, even with a substantial optimization, neither approach can guarantee a high yield of a capped and methylated mRNA precursor (J. Gen. Virol., 91:112-121, (2010)). Further, methods for preparing mRNAs with a Cap 2 structure are even more difficult and results are less predictable. Enzymes for converting Cap 1 to Cap 2 are not currently commercially available.
Another significant complication of in vitro synthesis of mRNAs, especially in large scale manufacturing, is the necessity to isolate and purify the active mRNA molecules carrying Cap from all uncapped mRNA forms which are inactive and in some cases immunogenic. Unfortunately, these methods are not trivial and often require a synthesis of modified mCAP analogs with conjugated affinity tag moieties allowing for easier isolation and purification of capped RNA transcript. Methods of synthesizing mCAP analogs with affinity tags as a reporter/affinity moiety and novel protocols for isolation of capped RNA from the transcription reaction mixture are known in the art (see, for example, U.S. Pat. No. 8,344,118). While these approaches are efficient, they require use of more expensive mCAP analogs and they allow for preparation and isolation of mRNAs containing the Cap 0 structure only.
The in vitro synthesis of natural and modified RNAs find use in a variety of applications, including ribozyme, antisense, biophysical and biochemical studies. Additionally, capped mRNA transcripts are used for applications requiring protein synthesis such as in vivo expression experiments (using microinjection, transfection and infection), in vitro translation experiments and assays as well as various applications in therapeutics, diagnostics, vaccine development, labeling and detection.
Consequently, there is a need in the industry for compositions and methods that allow for large scale synthesis of mRNAs that are (a) less laborious than conventional methods, (b) eliminate or reduce bi-directional initiation during transcription, (c) result in higher yields of mRNA, at a (d) reduced cost compared to current methods, (e) reduces production of heterogeneous products with different 5′-sequences and (f) does not require additional enzymatic reactions to incorporate Cap 1 and Cap 2 structures into the synthesized mRNA. There is also a need for the synthesis of various mRNAs containing modified and/or unnatural nucleosides, carrying specific modifications and/or affinity tags such as fluorescent dyes, a radioisotope, a mass tag and/or one partner of a molecular binding pair such as biotin at or near the 5′ end of the molecule.